Glioblastoma (GBM), the most common of the primary malignant brain tumors, is uniformly fatal. In contrast to current therapy, which is limited by off-target toxicity, immunotherapy promises an exquisitely precise approach, and substantial evidence indicates that, if appropriately redirected, T cells can eradicate large, well-established tumors. We have developed a bispecific antibody that effectively redirects T cells to lyse tumor cells expressing a tumor-specific mutation of the epidermal growth factor receptor (EGFRvIII) and found that our molecule localizes intracranially (IC) following systemic administration, mediating specific anti-tumor immune responses that eradicate well-established and invasive IC tumors. Preclinical models designed to test BiTEs to date, however, have universally employed xenograft systems. Such models are not ideal as they (1) do not account for proper evaluation of autoimmune toxicity, (2) do not fairly assess efficacy and the impact of a potentially suppressive tumor microenvironment, and (3) do not permit evaluation of potentially beneficial endogenous host immunity. In this proposal, we will conduct experiments employing a novel syngeneic system that directly addresses the clinically relevant limitations outlined above. Additionally, unlike other macromolecules, our data suggest that BiTEs may employ a unique mechanism to penetrate the blood-brain barrier (BBB), which may ultimately increase the biodistribution of the drug at tumor sites in the brain and have far-reaching implications for other therapies where BBB physiology is relevant. We therefore include studies to further our understanding of BiTE and T-cell biodistribution and will apply this knowledge to enhance the efficacy of the BiTE platform against IC tumors. Our overall goal is to further my training as a physician-scientist pursuing a career in translational neuro-oncology research while advancing the BiTE therapeutic platform for safe, effective immunotherapy in patients with EGFRvIII-expressing GBM. The completion of these aims will provide a strong foundation in basic science research as well as exposure to processes necessary to translate an experimental therapy to the clinic.?